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ATPase Assay
Make MTs as described elsewhere in protocol, using only unlabeled tubulin, spinning at 80k for 10 min in ultracentrifuge and resuspending the pellet in assay buffer + taxol. (Make sure DTT is fresh) # Measure concentration of tubulin by nanodropping for A280. Add 4ul of 6M Guanidine HCl to 1ul of resuspended MT. Blank with 4ul of 6M Guan HCl and 1ul of buffer. Read a few measurements and average. To get concentration in uM: (A280)*(Dilution1:5)*(10^6)/(115,000) ->115,000 is the extinction coeff of tubulin Ex: (0.56)*(5)*(10^6)/(115,000) = 24.3uM Boom, now you’ve got MTs. The assay is based on a reaction in which the regeneration of hydrolyzed ATP is coupled to the oxidation of NADH. Following each cycle of ATP hydrolysis, the regeneration system consisting of phosphoenolpyruvate (PEP) and pyruvate kinase (PK) converts one molecule of PEP to pyruvate when the ADP is converted back to the ATP (see figure). The pyruvate is subsequently converted to lactate by lactatedehydrogenase (LDH) resulting in the oxidation of one NADH molecule. The assay measures the rate of NADH absorbance decrease at 340 nm, which is proportional to the rate of steady-state ATP hydrolysis. The constant regeneration of ATP allows monitoring the ATP hydrolysis rate over the entire course of the assay. 3. Make all reagents. Assay Buffer: (Buffer of choiceBRB80, BRB12), 0.01% Triton X-100, 1uM DTT, 0.1mg/ml BSA. *Buffers and Triton X-100(presence or absence) and desire for any additional reagents are to be tested for optimal conditions. NADH: To obtain 5mM NADH: 1mg NADH(in deli fridge) + 282ul H2O PEP: To obtain 100mM PEP: 3mg PEP(in -30) + 133.4ul H2O + 11.6ul 5N KOH This PEP solution should have pH of 6.8. PK/LDH: Stock solution(in -30) has LDH at 900-1400 U/ml and PK at 600-1000 u/ml MgATP: Stock solution at 100mM Keep these 4 reagents on ice, and make NADH and PEP fresh before each session, NADH degrades quickly. 4. Usual concentrations during assay: MgATP (2 mM), NADH (0.1 mM), phosphoenol pyruvate (2 mM), pyruvate kinase (0.01 U), lactate dehydrogenase (0.03U). Again, these values are subject to change, and should be optimized if needed. 5. Now blank instrument with ATPase buffer Then mix in the following order: a. 45-x-y ul Assay Buffer b. x uL MTs to reach desired concentration c. 2 uL NADH d. 1 uL PEP e. 1 uL ATP f. 1 uL PKLDH g. y uL dynein/kinesin to meet good working concentration Mix well, then run assay (5 min reading time should be sufficient) Using the BioSpectrometer Basic -> Simple Kinetics -> BA INST This method has wavelength at 340nm, instant read, 37 degree celsius temper, 5 second intervals, 5 minute length read, instant read. Cuvette length 10mm, using the 50ul UVettes the long direction. Measure the decrease in 340 absorbance overtime, the machine records A/min. The machine records it as a negative value, in equations we use the change in absorbance, and therefore the magnitude, a positive value. To edit the points from which the slope is measured, go to process results and press linear regression, from here, use the arrows to move the first and last point of the slope. For basic kinetic analysis: ATPase Rate in mM/min = (rate of A340 lossA/min)/(pathlength(cm)*(6.23mM^-1cm^-1)) Molar Activity in 1/min = ATPase Rate/of ATPase enzyme Ex: if A/min = 0.9 for 100nM of Dynein. Molar Activity = ((0.9)/(6.23mM))/.0001mM = 1444.6 (1/min)